System for portable and easy-to-use detection of analytes with mobile computing device

ABSTRACT

This system takes in raw cellular material collected using a provided swab, blood collection device, urine collection device, or other sample collection device and transforms that biological material into a digital result, identifying the presence, absence and/or quantity of nucleic acids, proteins, and/or other molecules of interest.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 16/777,344, filed Jan. 30, 2020, which is a divisionalapplication of U.S. patent application Ser. No. 15/785,394, filed Oct.16, 2017, now U.S. Pat. No. 10,589,267, which is a continuationapplication of U.S. patent application Ser. No. 14/205,146, filed Mar.11, 2014, now U.S. Pat. No. 9,789,483, which claims the benefit of andpriority to U.S. Provisional Application No. 61/776,254, filed Mar. 11,2013, entitled SYSTEM FOR PORTABLE AND EASY-TO-USE DETECTION OF ANALYTESWITH MOBILE COMPUTING DEVICE, each of which are hereby incorporated byreference in their entireties.

FIELD OF THE INVENTION

The present disclosure relates generally to technologies for identifyingthe presence, absence and/or quantity of nucleic acids, proteins, and/orother molecules of interest within a sample.

BACKGROUND

Conventional technologies for identifying the presence, absence and/orquantity of nucleic acids, proteins, and/or other molecules of interestwithin a sample often require expensive laboratory equipment and theexpertise of highly-trained medical professionals. Consequently, suchanalyses are typically performed within laboratories or medicalfacilities. Such molecule detection can be important, for example, todetect the presence of pathogens, disease, contamination, overdoses, andpoisonings within an individual or other animal or the environment.Unfortunately, today, individuals may face long waits before the propertests can be performed and the results can be generated and analyzed.

SUMMARY

There is a significant need for improved molecule detectiontechnologies. Various embodiments disclosed herein may such a need.

The disclosed system takes in raw cellular material collected using aprovided swab, blood collection device, urine collection device, orother sample collection device and transforms that biological materialinto a digital result, identifying the presence, absence and/or quantityof nucleic acids, proteins, and/or other molecules of interest. Thesystem has several innovative components and subsystems to achieve theresult.

Overall, the architecture of the system includes a reader component, acartridge component that fits into the reader, a sample collectioncomponent that fits into the cartridge in the reader, and also a mobilecomputing device, such as but not limited to a smartphone or tablet PCsuch as an iPad®. Preferably, the reader communicates with the externalmobile computing device through wireless communication, especiallyBluetooth® protocols.

The mobile computing unit uses an App, or software application, to sendand receive signals with the reader, including new testing protocols,tests results, and more. The ability to add testing protocols hassignificant advantages for the system because this allows for sameonboard reader hardware to execute tests with new cartridges releasedafter the reader is already produced and in the hands of the user.

The mobile communication device allows for communication of the resultto physicians, recording of the result, and other options as describedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are described below with reference to theaccompanying drawings. In the drawings:

FIG. 1 depicts one embodiment of a system for detecting analytes.

FIG. 2 depicts another embodiment of the system.

FIG. 3 depicts an exploded view of one embodiment of a reader.

FIG. 4 depicts an exploded view of one embodiment of a cartridge. Notethat the actual component breakdown of the cartridge is on FIG. 5 . Thisfigure should not be used as a reference for component parts, but ratheras an example of how the cartridge stacks together into one unit.

FIG. 5 depicts an overview of the cartridge.

FIG. 6A-B depict an example of cartridge loading into the reader.

FIG. 7A-C depict an embodiment of the sample input mechanism.

FIG. 8A-D depicts one embodiment of inserting a swab into the cartridge.

FIG. 9A-B depict various views of a sonicator.

FIG. 10A depicts one embodiment of a sonicator at various times afterswab entry.

FIG. 10B schematically represents components present inside thereservoir.

FIG. 11A-C schematically represents the physical and chemical changesthat result from sonication.

FIG. 12 schematically represents a nucleic acid sandwich complex.

FIG. 13A-B depicts one embodiment of the reservoirs of the cartridge,without and with a swab, respectively.

FIG. 14A-B depicts a portion of one embodiment of a cartridge, whichincludes valves, with a first valve closed and open, respectively.

FIG. 15A-C depicts various views of one embodiment of a PCB component ofa cartridge.

FIG. 16 depicts one embodiment of a microfluidic channel and reservoircomponent with one embodiment of a swab.

FIG. 17A-C depict the progression of flow in one embodiment.

FIG. 18 depicts completed flow in one embodiment.

FIG. 19 depicts a top view of one embodiment of the microfluidic channeland reservoir component.

FIG. 20A-B depicts perspective views of one embodiment of themicrofluidic channel and reservoir component, before and after amembrane is placed and bonded on the component.

FIG. 21 depicts one embodiment of sensors with microbeads localized overit; a schematic representation of such microbeads is also provided.

FIG. 22A-B provides schematic representations of one embodiment of thesurface chemistry; one embodiment of microbeads are also schematicallyrepresented with and without a coupled target.

FIG. 23 illustrates the internal components of a one embodiment of areader, including an electrochemical circuit; a block diagram of theelectrochemical circuit components is also provided.

FIG. 24 depicts various views of one embodiment of an App.

FIG. 25A-B depicts a top view and a side view, respectively, of oneembodiment of a cartridge designed for multiplexing.

FIG. 26 depicts a side view of another embodiment of a cartridgedesigned for multiplexing.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The system disclosed herein takes in raw cellular material collectedusing a provided swab, blood collection device, urine collection device,or other sample collection device and transforms that biologicalmaterial into a digital result, identifying the presence, absence and/orquantity of nucleic acids, proteins, and/or other molecules of interest.The system has several innovative components and subsystems to achievethe result.

Overall, the architecture of the system includes a reader component, acartridge component that fits into the reader, a sample collectioncomponent that fits into the cartridge in the reader, and also a mobilecomputing device, such as but not limited to a smartphone or tablet PCsuch as an iPad. The overall architecture is shown in FIGS. 1-4 .Preferably, the reader communicates with the external mobile computingdevice through wireless communication, especially bluetooth protocols.As shown specifically in FIG. 1 , the system 100 consists of a durableuniversal reader and a disposable cartridge and swab. Communicationbetween a smartphone and reader occurs through Bluetooth 4.0 (BluetoothLow Energy), for example. Operating the device at a high level involvesthe following: 1) the user loads the cartridge into the reader, 2) swabshis nose and inputs it into the cartridge, 3) the test is run from amobile app (iPhone), and the results are displayed in the app as well.

The mobile computing unit uses an App, or software application, to sendand receive signals with the reader, including new testing protocols,tests results, and more. The ability to add testing protocols hassignificant advantages for the system because this allows for sameonboard reader hardware to execute tests with new cartridges releasedafter the reader is already produced and in the hands of the user.

The mobile communication device allows for communication of the resultto physicians, recording of the result, and other options as describedherein.

Cartridge Loading

The system is operated by first loading the cartridge into the reader200 as in FIG. 3 . The cartridge 300, as shown in FIG. 5 , includes: acover 310, which contains part of a swab input tunnel; a microfluidiccomponent 320, which includes reservoirs, part of the swab input tunnel,an internal membrane for swab input, area for an absorbent pad, amicrofluidic channel, and holes for wax valves over vias; a base 330with a magnet cutout/slot 332 for sliding a permanent magnet into placeduring cartridge loading; the base 300 also has a cutout 334 forsonicator access to the reservoir; a PCB 340 with sensors and valves andsonicator slot 344; and a swab 350 where the top is the head for samplecollection and input, and the swab has a neck for sealing. The cartridgecontains a slot to allow for the permanent magnet or magnets to slideinto place underneath the sensor component (labeled 332 in FIG. 5 ).This allows for the magnet to be as close to the sensor as possible,allowing for a greater magnetic field to be exerted, which is preferableto be able to utilize smaller magnets in bead capture (as described inanother section) with less overlapping fields or “crosstalk” of magneticfields.

The cartridge establishes electrical connections with the reader. In oneembodiment, this can be done with the leads shown at the top of thesensor component plugging into what is known as an “EDGE card”, aconnector with pins to establish electrical continuity. Theseconnections establish electrical continuity between the electrodes onthe sensor component and the electrochemical circuitry on the reader.Additionally, the resistive heaters on the sensor component establishelectrical continuity with the circuitry to establish current flowthrough the resistive heaters for valve actuation as described in thevalve section. Other connector schemes can be employed to achieveelectrical connections between the sensor component of the cartridge andthe reader.

The sensor component and the base of the cartridge also have a cutout topermit access of the sonicator piece to the reservoirs, particularly thesample preparation/input reservoir (see FIG. 5 ). When the user slidethe cartridge in, the cutout allows for the sonicator to be positioneddirectly underneath the reservoir. With the cutouts in the sensorcomponent and base component of the cartridge, the sonicator componentcan directly access reservoir 1 on the microfluidics component.

Underneath the reservoir, a high water content blister can optionally beaffixed in the cartridge production process such that the sonic energyconducted from the piezo component can be delivered with minimalattenuation into the reservoir during testing. This blister, or otherappropriately conducting sonication medium, is preferably dry on theoutside, with no liquid residue left behind. When the cartridge slidesinto the reader, the sonically conducting medium affixed to the piezodisc forms a soft seal with the sonically conducting medium affixed tothe bottom of the reservoir. This “soft seal” is enhanced by using aconformal sonically conducting medium on the bottom of the reservoir.

Additionally, the side of the reader can optionally have a pressuresensitive piezo electric component for sensing flex in the sample inputreservoir. This allows for a pressure sensitive start to the test assoon as a sample input device has entered into the reservoir.

To turn on the reader and to prompt a connection to the user'ssmartphone, the piezo cartridge has been inserted. Alternatively, theelectrical connections between the cartridge and reader can perform thefunction to turn on the reader, alert the reader to the presence of aninserted cartridge, and/or search for and connect to nearby smartphonesthat could be the users.

This particular function described in the preceding paragraph can beactuated by simply allowing a circuit to be completed with the additionof the cartridge based on electrical continuity between the reader andthe cartridge, just the same as a switch turns on an electrical circuit.

A resistor can be added to the sensor component that allows for thereader to distinguish between cartridges. In other words, if a cartridgeis added with a small surface mount resistor, or a resistive-ink basedresistive element, the reader can “read the resistor” using circuitry onthe reader. If the cartridge is recognized, the reader will prompt toopen an App established for this particular cartridge and/or a protocolon the App designated for this cartridge. If the resistor is notrecognized as a currently available cartridge, then the user will beprompted to either update the App designated to run the test, ordownload the proper App for running the test.

A default App can have a stored list of values for different possibleresistor values that helps facilitate locating the proper App to run thetest.

Once the proper App is located, the proper testing protocol will beadded to the reader's list of supported tests associated to theparticular resistance value of the cartridge. In other words, the newtesting protocol is loaded through wireless communication (i.e.bluetooth) onto the reader so that future tests with this cartridge willautomatically be recognized and performed without the need for searchingfor a new protocol.

An example of cartridge loading is provided in FIGS. 6A-6B. In FIG. 6A,we are looking at the reader from above. The top is removed for theview. As shown in FIG. 6B, the user loads the cartridge into the reader.The cartridge establishes electrical connections with the reader via anedge card connector. The permanent magnet slides into place beneath thesensors for magnetic bead capture. This sliding into place happenswithout bumping into the cartridge—made possible by the cutout in thebase of the cartridge, and also allows for close proximity of the magnetto the microfluidic channel to attract beads onto the sensor. The cutoutin the base and the sensor component allows for a piezo to slide intoplace underneath the reservoir for sample preparation.

Leakage-Free Rupture of Internal Membrane

This sections figures are FIGS. 7-9 . The user pushes the swab into theplastic cartridge and the swab locks into place automatically. The swabhead ruptures an internal membrane containing a volume of liquid. Theliquid allows for material collected on the swab to be suspended in thefluid for analysis, as described in follow sections.

In FIGS. 7A-C, a sample input mechanism is shown. In FIG. 7A, thecartridge contains a swab input tunnel. At the end of the tunnel, wehave a membrane covering the entry port to the first reservoir. This iswhat we call the internal membrane. FIG. 7B shows the swab travelingthrough the swab tunnel (the cover is made invisible here) towards thearea for the internal membrane 750. FIG. 7C shows the swab piercing themembrane and entering the first reservoir.

In the example of FIG. 8 , the cartridge 300 includes a membrane 322, afirst reservoir 324, a second reservoir 325, and a third reservoir 326.The reader 200 includes a sonicator 250. In FIG. 8 , at FIG. 8A, whenthe user inserts the swab into the cartridge 300, the swab head pushesthe membrane 322 in, as shown at FIG. 8B. It then ruptures the membrane322, right as the neck of the swab takes up position where the membrane322 was located to seal off the reservoir 324 and prevent leakage, asshown at FIG. 8C-8D. The swab head is located in reservoir 324, directlyover the sonicator 250, as depicted. Inside this reservoir 324, we havea volume of liquid that contains several components as discussedelsewhere herein. (Note that the cover for the microfluidics, whichmakes up the top of the swab input tunnel, is not shown in FIG. 8 ).

The swab head is meant to rupture an internal membrane, yet there aretwo reasons the swab head is blunt:

-   -   (a) the user should not be able to hurt themselves with a sharp        swab head    -   (b) in our approach to rupturing the internal membrane, it is        critical that the swab is blunt enough that it doesn't        immediately pierce the membrane. Instead, it continuously        deforms the membrane to the rupture point.

Two critical events happen at the instant the swab pushes the membraneto its rupture point:

-   -   a) The rubber gasket at the base of the swab head has moved into        position to form a seal with the structural material surrounding        the membrane.    -   b) The swab shaft is far enough inside the plastic cartridge's        tunnel that the swab locks into place. The locking into place is        critical to maintain some structural support for preventing        leakage. In particular, the swab being locked into place allows        the swab seal to resist the pressure exerted on the head during        rupture of the membrane.

Every material has a modulus of elasticity, which is a constant thatallows a calculation of when a material retains its elasticity, i.e. howmuch the membrane can be stretched yet will return to its originalshape. This point is called the yield point. Beyond the yield point, thematerial exhibits plasticity rather than elasticity, where it deformsand no longer returns to its original shape. Beyond the yield point isanother critical point called the rupture point. The rupture point iswhen the membrane breaks. If instead the swab head was sharp or themembrane material had a very small rupture point, the swab would quicklypierce the membrane, making it very difficult to contain the liquid asit spilled from the membrane. A blunt swab with an appropriatedeformable membrane allows the swab's gasket to get into place toprevent leakage is key. Elastic membrane materials are readily obtainedfrom polyurethane, polysilicone and polybutadiene, and nitrile forexample. Deformable, inelastic diaphragms are made with polyethyleneterephthalate (PET), mylar, polypropylene, polycarbonate, or nylon, forexample. Other suitable materials for the deformable film includeparafillm, latex, foil, and polyethylene terephthalate. Key factors inselecting a deformable film include the yield point, rupture point, andelastic modulus.

The size of the swab head and the rupture point of the membrane materialand the location of the internal grooves must be decided inconsideration of each other.

Swab Locking and Clicking into Place

The swab clicks or locks into place because of matching positive andnegative grooves in the tunnel. The positive grooves are radially placedin the tunnel and negative grooves are placed on the shaft of the swab.They are formed such that when the swab shaft moves past them they arecompressed slightly, but as soon as the swab is in the right location,they lock into the matching groove on the swab. The reason for thisstructure is three fold:

-   -   (a) there should be tactile confirmation for the user that the        swab was inserted correctly to help them ascertain the proper        depth of swab input    -   (b) the two-way lock gives structural support to the        rupture/seal mechanism    -   (c) the two-way lock prevents the user from taking the swab out        of the cartridge    -   (d) radial placement allows for non-oriented insertion of swab        by the user; the negative groove on the swab can be just a        single groove along the circumference of the shaft.

By two-way lock, we mean that the positive and negative grooves are deepenough that they prevent the user from both 1) being able to push theswab in further and 2) pull it out. Preventing the user from taking outthe swab is important because this allows for the easy disposability ofthe system into normal trash and also prevents the user from coming intocontact with contaminated items. This is a significant advantage from abiohazard trash perspective.

Pressure Sensitive Auto-Start to Test

Once the cartridge is loaded into the reader, we need a 1-step methodfor starting the test. In other words, we would like for the user to beable to input the sample input device, such as the swab, blood lance,saliva collector and have the test start upon inserting this inputdevice into the cartridge without having the user required to push abutton. This can be accomplished with the following method.

The reservoir accepting the input is made to flex slightly such thatupon the input devices entry into the reservoir, the flex outwardoccurs. A pressure sensitive sensor on the reader, such as a piezoelectric sensor, in contact with the reservoir then reports the pressurechange to the processing unit on the reader, which in turn begins thetesting protocol.

For example, as shown in FIG. 9A, the swab insertion, which moved theswab passed the membrane area 910 into the first reservoir 915, hasbrought the head containing the sample directly over the sonicator 920.The sonicator 920 directs all of its energy into this first reservoir915 to mix the sample on the swab head with the reagents. As shown inFIG. 9B, the sonicator piece consists of a piezo disk 922, a piece ofmaterial called Aqualene 924 that conducts high frequency sound wavesand a cup 926 for holding the parts together. Other sonically conductingmediums can be appropriate. Additionally, on the underside of thereservoir, a sonically conducting medium can be affixed such that a softseal is formed between the conducting medium permanently affixed to thepiezo component and the reservoir.

The pressure sensitive piezo and the flex region of the cartridge can beanywhere along the swab input tunnel, as long as the change in pressureoccurs as the swab head actually inserts into the input.

Upon receiving the increased pressure and upon conversion into a signalthe microcontroller on board can read as a test initiated signal, themicrocontroller will start the testing protocol and send notificationvia wireless communication to the mobile computing unit (i.e. iPhone®)and suggest to open the App, while maintaining a timer than can be usedto update the iPhones progress bar for test completion percentages andestimated time to completion.

The key points are for the reservoir, or other area along the inputtunnel to flex outward increasing the pressure on a pressure sensor onthe reader in contact, which will then initiate the testing protocol andpossibly inform the remote, mobile computing device (smartphone, tablet,etc.).

Other Sample Collection Devices

Swabs are not the only sample input device that can be used to inputsample into the cartridge. Alternative collection devices, such as onethat wicks a small droplet of blood or urine into a small capillarychannel can be used to input collected sample into the reservoir aswell. Throughout this document, swab head will be the term used toreflect the sample collection portion of any sample input device.

Sample Preparation

This sections figures are FIGS. 9-14 . At the input reservoir (where theswab head enters), a liquid volume is present to recover sample on theswab head. This material, be it nasal sample, blood sample, etc.potentially has an analyte of interest to be tested. Therefore, in thisinput reservoir, there are several important components to accomplishthe key objective of the sample preparation phase: to assemble asandwich complex onto one or more populations of microbeads ornanoparticles using antibody probes or nucleic acid probes or otheraffinity reagent as depicted in FIGS. 10 b and 12.

The components in the liquid reservoir needed to form the sandwichcomplex include the (a) components that actually make up a bead-basedsandwich complex: microbeads/nanoparticles with surface bound affinitymolecules (antibodies, nucleic acid probes, etc.), detector agents suchas antibodies conjugated to signaling enzymes, labeled nucleic acidprobes to which signaling enzyme can bind; (b) agents that facilitateformation of the sandwich complex such as salts; (c) agents thatfacilitate access and specificity to target analytes such as detergentsand enzymes for lysis or chopping up larger nucleotides into smallerpieces; (d) blocker proteins to decrease nonspecific binding ofsignaling enzyme or targets onto microbeads and sensors downstream; (e)stabilizers such as trehalose that can keep the components in thereservoir functional throughout a long shelf life.

Components that make up a bead-based sandwich complex include:Microbeads/nanoparticles, and there can be multiple populations ofthese, each with their own affinity (in other words a different antibodyor set of antibodies or dna probes per population of bead), signalingenzyme, “detector” antibody or another antibody or nucleic acid that hasaffinity to another portion of the analyte of interest and a signalingenzyme, such as horseradish peroxidase or soybean peroxidase. Thesandwich complex is well known in ELISA and there are multiple possiblecombinations to form such a complex. A common one is depicted in FIG.11B-C for antibodies detecting proteins or small molecules of interestand FIG. 12 for nucleic acids with sequence specificity. Additionallynucleic acid aptamers could be the affinity unit on the surface of thebead or other affinity molecules. As shown in FIG. 11B, if target 1101is present, the capture antibody will grab the target 1101 and thesignaling antibody will also grab hold of the target, forming a sandwichcomplex. This signaling antibody's attached HRP enzyme will then cause asignal to occur at the sensor downstream. As shown in FIG. 11C, iftarget 1101 is not present, the signaling antibody/enzyme will notattach to the bead and will not form a complex with the bead anddownstream no signal will be generated over the sensor.

The example nucleic acid sandwich complex of FIG. 12 includes: a microbead 1201, probably magnetic; a capture probe 1202 fixed to the surfaceof the bead 1201, it being a DNA nucleic acid probe; a single strandednucleic acid target 1203, either RNA or DNA—note that this piece hasmore bases than either the capture probe 1202 or a detector nucleic acidprobe 1204 such that the capture probe 1202 can hybridize to a portionof this target 1203 and there still be bases available for hybridizationfor the detector nucleic acid probe 1204, which possesses the detectorcomplex; a detector nucleic acid probe 1204 labeled on the end distantfrom the bead 1201 such that it can host an hrp enzyme 1205; and asignaling enzyme 1205 can be bound to a detector nucleic acid probe 1204through common labeling scheme affinity such as: HRP conjugated tostreptavidin binds to biotin-labeled detector probe 1204.

The possibilities for forming the complex are endless and include usinga biotin labelled antibody which binds to a portion of the target,antibodies and nucleic acids can both be pre-biotinylated such that astreptavidin conjugated signaling enzyme such as HRP can then bind thebiotinylated detector to form a complex. This confers a target specificbinding of HRP onto the bead and is quantitative to the amount of thetarget captured. The label combination is of course not limited tobiotin-streptavidin. Any suitable labeling scheme will work.Additionally, multiple HRP enzymes can be conjugated together into amolecule commonly known as a Poly HRP molecule such that the signalgenerating capability of a sandwich complex can be enhanced.

Salts are necessary in the input reservoir to enhance the likelihood ofbinding. There are some salt combinations that will interfere withelectrochemical detection downstream, but phosphate buffered saline isan appropriate solution with the right salts to contain all of thecomponents in.

Blocker proteins, such as the well-known Bovine Serum Albumin, Casein,Fibrinogen, etc. that help to stabilize other proteins such asantibodies and enzymes, but also help to prevent non-specific binding ofHRP or other signaling enzymes to the beads and to the channel walls (inthe microfluidic system described later) are used in the system as well.

Additionally, for assays that require lysis, detergents are oftenrequired. Nonionic detergents, rather than ionic detergents, arenecessary as to not denature the signaling enzyme or antibodies in thesolution. Detergents can enhance lysis of bacteria, but are also usefulfor gently lysing various viruses, such as the influenza virus. This isdesirable to improve access to targets such as nucleoproteins that areinternal to the virus.

Enzymes that enhance lysis and reduce viscosity during lysis are also anecessary component in the preparation of samples containing bacteria,for instance E. coli. The enzymes that facilitate lysis includelysozymes and DNAses that do not disrupt the probes on the surface ofthe microparticle but do chop up genomic DNA released are useful forpreventing severe increases in viscosity that hamper bead movement.

Enzymes such as RNAses or DNAses that selectively chop larger nucleotidesequences into smaller sequences can be useful for generating smallerfragments that show favorable binding kinetics.

Another component is a stabilizer agent such as trehalose, which helpsprotect proteins from oxidation and therefore increases the shelf-lifeof the solution, especially at room temperature.

With the sample collection device entering the sample preparationreservoir, a key factor is the placement of the sample containingportion (the swab head) directly over the sonicating component as inFIG. 9 . The PCB sensor board component of the overall cartridge musteither have a cutout or otherwise not extend to the reservoir region inorder to allow direct access of the sonicator unit to couple to thesample preparation reservoir. The bottom of the reservoir can also havea sonically conducting material that can form a soft seal with the sonictransmitting material directly over the piezo disc. This forms acontinuous channel for the sonic energy to transmit into the reservoirfrom below all the way from the piezo disc.

The first phase of this sample preparation process is a high intensitysonication to achieve proper suspension of the reagents, particularlythe beads in the reservoir such that these beads are available insolution to capture the target which is actively being eluted into thereservoirs solution with the aide of the sonication. An appropriatesonication piezo is a 1.6 mhz bending transducer piezo. The point isthat it is not meant to generate cavitation and large shearing forces,but rather a gentle sonication, even at the high intensity phase.

Then the sonication is pulsed in order to keep the beads from settlingand to continue to add energy into the system to enhance thehybridization between the affinity reagents on the bead and the targetand the signaling antibody or nucleic acid for selective enzymeretention. Refer to FIGS. 10 a and 11.

As shown in FIG. 10A, after a few seconds, the sonicator helps to mixthe sample with the reservoir's contents. FIG. 10A(i) shows the swabjust after it is inserted. FIG. 10A(ii) shows the reservoir secondsafter the sonication protocol is initiated. In FIG. 10A(i), the red dyefrom the swab is not mixed in the reservoir 1002; in FIG. 10A(ii), thered dye has mixed fully within the reservoir 1002. As represented inFIG. 10B, inside the depicted reservoir, we have a mixture containingmillions of magnetic microbeads 1010 or nanoparticles possessing captureantibodies 1012 on their surface very specific to target proteins; thesetarget proteins can be components on a viral membrane (for detection ofviruses), proteins such as CRP, or even small molecules such ascaffeine. Also cohabitating the reservoir is the signaling antibody1014, which is conjugated to an enzyme 1016 called HorseradishPeroxidase (HRP) that can oxidize a chemical substrate (which means itstrips it of an electron). This electron stripping can be measured onthe electrochemical sensor and is proportional to the amount of targetpresent. A more complete list of components is listed elsewhere herein.

FIG. 11A illustrates that in the reservoir 1100 there is a settled phaseand an excited phase. In the settled phase, before sonication, beads aresettled in the reservoir 1100. In the excited phase, during sonication,beads are mixing with the swab's payload (not pictured: the swab head).This allows for capture of target molecules.

The sonication profile, in other words, how long the piezo is on for andwhether it is pulsed etc. varies according to the sample being tested.It is preferable that the system has fine grain control over thesevariables. In particular, for power consumption purposes, it ispreferable that the piezo has an “on period” in which the piezo“pulses”. In other words, for every 10 seconds the piezo is on forexample 3 seconds and within that 3 seconds, the piezo turns on for0.027 seconds and turns off for 0.027 seconds. These methods allow forthe sample to not get too hot, to allow for hybridization, and to avoidconsuming too much power, important in a battery operated system, whilestill providing the environment conducive to target capture and sandwichcomplex formation. See figure x for a screenshot of variable controlover the piezo.

Another innovative feature of our system is the ability to update thesonication profile depending upon the cartridge and sample being tested.In other words, we utilize the remote updatability of the App, whichcommunicates with the reader, to command and control the piezo such thatit provides a different sonication profile depending upon the cartridgebeing added. Since more cartridges and tests are added to the systemover time, and since all of the profiles don't have to be preloaded, itallows for much more flexibility in the system for handling newcartridges/tests.

Now that the beads have captured target (if present) and formed thesandwich complex, the system utilizes the liquid medium used for formingthe complex as a transportation medium for getting the beads to thesensors by capillary flow of the whole solution past the sensor ofchoice.

The method:

-   -   (1) the system opens a valve to allow the content of        input/sample preparation reservoir (hitherto designated        reservoir 1) to flow out using    -   (2) Microfluidics (capillary flow) to transport beads from        reservoir 1 to the sensor, where a magnet underneath will        localize these magnetic beads directly over the sensor.    -   (3) Then the system uses the same valving and flowing mechanism        to wash away excess signaling enzyme not bound to any beads to        remove nonspecific signal (wash solution is preferably in        reservoir 3, most upstream)    -   (4) And then the system reuses the same valving and flowing        mechanism to provide the chemical substrate the enzyme needs to        create a net flow of electrons in proportion to the amount of        target present (chemical substrate is preferably in reservoir 2,        between reservoir 1 and 3.) This electron flow generated by        oxidation/reduction chemistry is what we will measure on our        electrochemical sensor.

The key here is to take the already formed sandwich complex bead (iftarget is present) and use the volume of liquid acting as a flow mediumin a capillary channel to transport and passively deposit the beads ontothe intended sensor.

The beads are magnetic, and therefore they are down from the transportsolution to the surface of the sensor via magnetic force generatedpreferably through a permanent magnet located beneath the sensor (aspart of the reader, not the cartridge), but this magnetic force can alsobe generated through electromagnetism from coils located on the sensorcomponent of the cartridge.

Valve

This sections figures are FIGS. 13-15 . The use of an inline productionprocess of printed circuit boards to create a valve actuating element ina diagnostic product is described here. A via is a standard product ofPCBs that is typically used for allowing signal traces on one layer of aPCB to continue electrically with another layer. The vias provideelectrical continuity through multiple layers. Our system utilizes thefact that they are also excellent conductors of heat to a very preciselocation without affecting the areas around it because the material thatcomprises most PCBs, such as FR4 are excellent insulators of heat. Thisallows for minimal “crosstalk” between valves located close to eachother.

The system utilizes these vias as point sources of heat to melt wax,preferably hydrophilic wax, that is holding back a volume of liquid. Aresistive heating element generates the heat that is to be conducted tothe exact location where the wax needs to be melted such that thereservoir has an opening to which its fluidic contents can drain throughthe opening into a microfluidic channel. This heating element can be onthe board, as in serpentine trace in FIG. 15 (on the backside of thesensor component), or can be external to the cartridge board. In otherwords, the heating element can be located on the reader, but with springloaded contacts to form an effective contact with the via, such that thevia can conduct the heat to the wax barrier.

Therefore, the innovation has a heat conducting via created on thesensor component of the cartridge, a resistive heating element, a waxbarrier, and a volume of liquid being held back. The wax forms a sealwith the hole in the reservoir to prevent liquid from leaking into themicrofluidic channel on the microfluidic component of the cartridge.

Current is allowed to flow through the resistive element, most likelythrough actuation of a transistor. Current passing through the resistiveelement generates heat through Joule heating. Because of physicalcontact between the resistive heater and the via, the heat is conductedthrough the via up to the wax barrier. Because of the heat, the waxbarrier melts, allowing the liquid volume the wax was holding back toflow past the location of the former wax barrier.

To create the valve (not actuate during system operation):

It is preferable if the wax has the minimum height necessary to occludethe opening between 1) reservoir of liquid held back and 2) themicrofluidic channel in order to minimize the distance heat must travelto melt the wax.

The preferred method for realizing this wax barrier is to apply meltedwax to a heated via area such that the wax does not freeze upon contactwith the via, which causes and unnecessary excess of height.

The valves are prepared by application of heat to the chip, especiallythe via area, such that the wax does not freeze on contact, which wouldcause an undesirable increase in height. Pancaking of the wax ispreferable to minimize the height, which will maximize the chance ofproper melting actuation of the valve. The heating is important becauseit allows for proper, regular, consistent formation of the valve.

The reservoirs preferably have an angled bottom such that dead volume isminimized. In other words, the area closest to the opening of thereservoir to the channel is at a lower height relative to the area ofthe reservoir further from the channel.

As shown in FIG. 13A, in the first reservoir 1301 occurs sample inputand sample preparation; in the second reservoir 1302, is an enzymesubstrate; and in the third reservoir 1303, is a wash solution. There isa valve opening 1304 at the base of each reservoir connecting to amicrofluidic channel 1305. FIG. 13B shows a swab head 1350 in the firstreservoir 1301.

In FIG. 14 , we use a phase change valve consisting of a hydrophilic waxwith melting temperature around 50 degrees Celsius. A small amount ofwax is deposited very precisely onto a via, which is continuous with aresistive heater on the bottom of the green sensor board (shown in FIG.15 ). The via acts as a very precise shaft for delivery of heat directlyto the location of the wax. This wax, in its solid state, seals thereservoir's opening that meets the microfluidic channel. When current isflowed through the resistive heater the via heats up the wax, meltingit. The seal is broken, allowing liquid to flow out of the reservoirinto the microfluidic channel. These valves are individually addressableand automated. Specifically represented in FIGS. 14A-B is a firstreservoir 1401 for sample input and preparation, a second reservoir 1402for enzyme substrate, and a third reservoir 1403 for wash solution. InFIG. 14A, the first valve 1411, the second valve 1412, and the thirdvalve 1413 to the microfluidic channel 1405 are closed. In FIG. 14B, thefirst valve 1411 is open, and there is flow. The second valve 1412 andthe third valve 1413 are closed.

FIGS. 15A-C show the sensors and valve vias on the PCB sensor componentof the cartridge. Specifically, FIG. 15A is a top view. The via 1505delivers heat to the wax barrier. FIG. 15B is a backside view showing aresistive heater 1510 (formed by serpentine trace). FIG. 15C illustratesthat an opening 1520 at the bottom of the reservoir meets the via, wherewax is deposited to form a seal, preventing leakage of liquid into themicrofluidic channel.

Flow

This section has reference FIGS. 16-21 . In FIG. 16 there is shown amicrofluidic and reservoir component with a swab. Depicted elementsinclude: a first reservoir 1601 for sample input and sample preparation,a second reservoir 1602 with enzyme substrate, a third reservoir 1603for wash solution, a microfluidic channel 1605, and an area for anabsorbent pad 1610.

Upon completion of the sonication/preparation protocol, the liquidvolume containing the beads becomes the transport vehicle for taking thebeads from the preparation reservoir to the sensor for detection.

The liquid from the first reservoir 1601 flows downstream of thereservoir towards the sensors. There must be a vent downstream of thereservoir to allow displaced air to vent as the liquid flows into thechannel 1605.

There must be a vent in the reservoir 1601 itself to allow for fluidflow (air) to replace the fluid (liquid) entering into the channel 1605.This can be formed by an air permeable PTFE membrane on the top of thereservoir component. The advantage of using PTFE is that it acts to sealoff the reservoir from leakage out the top, yet allows for the liquid todrain out of the reservoir during flow of liquids from the reservoirthrough the microfluidic channel.

The hydrophilic channel is constructed by the sensor component of thecartridge making one of the walls and the microfluidic component of thecartridge makes up the other 3 walls of the channel. This channel can bemade hydrophilic with the appropriate thermoplastic resin used to createthe piece, or via surface modification, especially via pegylation(polyethylene glycol) grafting to the surface of the walls mediated byplasma treatment to activate the walls such that the PEG will bond,making a hydrophilic and protein resistant surface.

Additionally, a lateral flow type membrane, commercially available maybe used such that the channels interior is not just empty space, but awicking material.

The reservoirs are sequenced in a particular order. The samplepreparation reservoir is the furthest downstream, closest to thesensors. The wash reservoir is furthest upstream. The chemical substratereservoir for enzyme reaction is between sample preparation and washreservoirs. The sample input reservoir is preferably first in order toaccommodate the sample input and it must be upstream relative to thewash reservoir such that all enzyme not bound to the target carryingbeads will be washed away and not create non-specific signal at thesensor. If the wash reservoir was downstream of the sample input, thenunbound enzyme would get pinned against the upstream-most vent, causingnon-specific signal to occur when the chemical substrate is released.This non-specific signal will drift downstream and be read by thesensors.

The valves are actuated in the sequence: sample preparation, wash, thenchemical substrate. A certain amount of time is allotted for each valveto actuate, then flow time for the reservoir to empty it's componentsinto the channel and be sucked up by the absorbent pad downstream of thesensors. Enough time for the absorbent pad to suck the channel dry suchthat very little to no fluid is left in the channel can be desirablesuch that very little mixing occurs between successive reservoirscontents.

At the end of the microfluidic channel there is an absorbent pad whichwicks liquid from the microfluidic channel, downstream of the sensors.The volume the absorbent pad can wick must be enough to drain the samplereservoir and the wash reservoir, but only enough to pull the chemicalsubstrate reservoir into the channel and over the sensors, then stopsuch that there is little to no flow over the sensors when enzyme boundto beads is turning over the chemical substrate to cause a detectablesignal over the sensors where they are electrochemically “read”.

The sample preparation volume of liquid transports the beads to thesensors where they are caught via magnetic force over a sensor fordetection. The channel is washed and chemical substrate is provided forany enzyme bound to beads.

An example of flow to transport beads to sensor is provided in FIGS.17A-C and 18. FIG. 17A shows flow 2 seconds after valve burst; FIG. 17Bshows flow 4 seconds after valve burst, and FIG. 17C shows flow 12seconds after valve burst. In FIG. 17A, when the sonication protocol iscomplete, the valve for the first reservoir is actuated and the contentsflow into the main microfluidic channel. In FIG. 17B, the absorbent pad1710 draws the liquid completely out of the reservoir. The magneticbeads are localized over sensor 1722 (which has a magnet locatedunderneath). In FIG. 17C, the reservoir is emptied, and now we will washthe channel with the volume of liquid contained in the third reservoir(clear liquid). Notice that sensor 1722 is darker than sensors 1721 and1723 because of bead localization. As shown, an ambient noise sensor canalso act as a control to determine the test was performed correctly.Sensors 1701 and 1703 are used to subtract out ambient electrochemicalnoise. In FIG. 18 , the flow is complete. The microfluidic channel 1705is filled with enzyme substrate from reservoir 1702. The channel 1705has now been washed with the contents of the third reservoir (empty);the chemical substrate from the second reservoir 1702 (empty) needed bythe signaling enzyme is now in the channel. Detection can now takeplace.

Bubbles are often a problem in microfluidic systems. To counter thisissue, at least some of the top of the microfluidic channel (on themicrofluidic component of the cartridge) is replaced with PTFE membranesuch that the PTFE forms the “ceiling” of much of the channel (as inFIG. 20 ). This allows for passive degassing of bubbles contained withinthe channel. The pore sizes of the PTFE membrane can vary and includeanything from 0.1 microns to 3 micron pores and the membrane can besealed onto the channel with adhesive.

FIG. 19 shows an example in which the top of the channel 1905 in themicrofluidic component is cut out. A lip 1906 on both sides of thechannel 1905 allows for bonding of PTFE membrane on top of the channel1905. A vent 1908 is also provided. In FIG. 20A, it is again visiblethat the top of the channel 1905 is cut out. Also visible is the PTFEmembrane 2000 (wide enough to bond to lip on both sides of the channel1905). In FIG. 20B, the PTFE membrane 2000 is in place and bonded toform a microfluidic channel top for passive degassing of bubbles.

Because PTFE is hydrophobic, prewetting is generally required for liquidto flow properly along the PTFE membrane. To prevent a distinctprewetting step, as opposed to just flowing the sample preparationreservoirs contents into the channel, a “structural prewetting” can beaccomplished.

What is meant by structural prewetting is that rails of hydrophilicmaterial can run the length of the PTFE membrane promoting flow ofliquid along the rails to facilitate the first reservoirs liquid to flowin the microfluidic channel. The hydrophilic rails help overcome thehydrophobic resistance of the PTFE. These rails can be formed in amultitude of ways that include thin plastic rails that span the lengthof the area where the PTFE acts as the ceiling of the channel. In otherwords, the PTFE will be covering the rails as it acts as themicrofluidic channel's ceiling. Additionally, adhesives directly on thePTFE can form the rail, or a patterned surface modification of the PTFEmembrane such that the surface modification for hydrophilicity run thelength of the channel.

Detection

The electrochemical sensors where detection takes place are preferablymade through ENIG process and thus have gold on the surface. There arethree electrodes, a working, counter, and reference electrode. Each havea surface chemistry formed onto them. This has thiolated ethyleneglycol, preferably a dithiol such as hexaethylene glycol dithiol foradded stability. The hydrophilic nature of the head groups facilitatesflow and protein resistance. The surface is preferably backfilled withmercaptoundecanoic acid, Another potential backfiller ismercaptohexanol. The layer is formed by sequential addition andincubation of first the ethylene glycol dithiol and then backfiller atunelevated temperatures.

Notably, the method does not require affinity reagents at the sensorsthemselves, which enhances the overall speed of the system.

Besides the target sensor, it is desirable to have an ambientelectrochemical noise sensor which detects background noise in thesystem from non-specifically bound enzyme. This background noise sensorwill then be used to help subtract out system noise from the signalgenerated at the target sensor.

As an example, as shown in the photograph and zoomed in schematicrepresentation in FIG. 21 , sensor 2102 has millions of microbeadslocalized over it. Excess HRP signaling enzyme has been washed away andchemical substrate for the HRP is the only liquid remaining in themicrofluidic channel. Note that while the beads are depicted assuspended over the sensor, in reality the magnet will localize the beadsagainst the surface of the sensor, not in suspension above it. Theenzyme substrate solution is in the channel containingTetramethylbenzidine molecules (from the second reservoir) and hydrogenperoxide; both are necessary for signaling enzyme to operate to create acurrent flow in proportion to presence of target. As shown, the workingelectrode 2104 of sensor 2102 has surface chemistry 2106: a carefullydesigned self-assembled monolayer reduces non-specific signal generationfrom enzyme sticking and smooths over defects on gold surface, leadingto much more reproducible results.

The detection is preferably carried out using standard electrochemicalcircuit that utilizes a bias potential generated at the referenceelectrode such that the oxidation/reduction reaction can proceed. Thepotential is held at the reduction potential of the chemical substrate(low enough that there is little nonspecific reduction of reduciblespecies in the solution) such that the flow of electrons to the oxidizedmolecules can be quantified using a current to voltage op amp connectedto the working electrode. For example a common substrate,tetramethylbenzidine is used for HRP. When present, HRP oxidizes TMBmolecules, and these molecules are in turn reduced by the workingelectrode. Since this event occurs in proportion to the amount of HRPpresent, we see a change in the current to voltage op amp that ismeasured by the analog-to-digital converter, which we can then take asthe actual signal to be interpreted by the microcontroller as depictedin FIG. 23 . As further shown in FIG. 23 , the electrochemical circuit2300 (shown amongst the reader internals) converts the current sensed atthe working electrode to a voltage using a current-to-voltage op ampscheme. This analog signal is routed to an Analog-to-Digital-Converter(ADC) on the microcontroller in the reader. From there, the signal istransmitted via Bluetooth 4.0 to the iPhone 2301, where the result isinterpreted and displayed.

An example of the App display is provided in FIG. 24 ; in the example,flu was detected. The App provides customized information based onpersonal medical history entered earlier (asthma for instance).Antivirals are a strong option for treating flu and recommended by theCDC. You can set pharmacy information in the app and transmit result todoctor with pharmacy phone number. You can let others in your networkknow (for instance other mothers at your daycare via Facebook).

HRP or other signaling enzyme is present over the sensor in proportionto the amount of target captured because of the formation of thesandwich complex to which it is a part and which can only be formed inthe presence of target. Refer to FIG. 22 . Specifically, FIG. 22A showsthe working electrode with target present, and FIG. 22B shows theworking electrode without the target. With the target: HRP, thesignaling enzyme, will oxidize the TMB molecules in the solution (fromthe second reservoir), causing a net flow of electrons into the cell.This flow of electrons is measured by the electrochemical circuitry. Theamount of signal is proportional to the amount of signaling enzyme,which is proportional to the amount of target. This gives us aquantitative result concerning the amount of target captured. Withoutthe target: there is not signaling enzyme to oxidize TMB, thereby littleto no flow of electrons into the cell. The surface chemistry is criticalfor preventing HRP signaling enzyme from sticking to the gold sensorsand producing non-specific signal.

The bottom of the cartridge has a cutout that allows for the permanentmagnets to be closer to the sensors as the cartridge slides in. Thecutout allow the cartridge to slide in without hitting the permanentmagnets. The closer the permanent magnets are to the sensor, the moreforce they are able to exert, meaning that smaller magnets are capableof exerting equivalent magnetic field strengths to larger magnets whichare more costly. Refer to FIG. 5 . Additionally, smaller magnetic fieldscan limit the amount of cross talk between magnets under differentsensors, which is important in a multiplexed assay, which is discussedin the Multiplexing section.

Multiplexing

A few innovative methods for achieving multiplexing are discussed. Theyall begin with the same initial premise: that initially multiplepopulations of beads exist in the first reservoir.

A population of beads is defined by having two differentiatingcharacteristics:

-   -   1) affinity to a certain set of targets (through capture        antibody, capture DNA probe, or other affinity molecule on the        surface of the bead)    -   2) a difference in size, magnetic response, density, or any        combination thereof

No two population of beads can have the same affinity (1) or samedifferentiating physical characteristic to be exploited for separation(2).

The beads are utilized to capture the targets as in the methods earlierdescribed. The key point is that a distinguishing physicalcharacteristic between bead populations allows for the differentiatedquerying of target acquisition (see detection section) since differentpopulations of beads have different target sets.

Method 1, Dead-End Filtration Through a Series of Size ExclusionFilters:

This method relies on differentiation of bead size. Dead-end filtrationis a commonly used term for filtering through a membrane. In this methodwe utilize the same principle in an innovative way in order to achieve amultiplexed and quantitative interrogation of multiple sized beads. Asthe beads flow out of the reservoir and into microfluidic channel, wehave a sequence of filters in order of largest pore size filter first.Each filter is placed in close proximity to an electrochemical sensorthat is designated to read that population of beads. Beads of smallersize than pore size will pass through the filter with the flow of liquiddown the channel. Beads larger than the pore size will remain behind, inclose proximity to the sensor designated to read its signature. Throughsuccessive filters, the bead populations in order of larger to smallerare trapped over the sensors designated to read them. In this manner,different populations of beads with different target sets are thenqueried in the manner we have described earlier in the detectionsection.

This process can be enhanced through use of magnetism. Beads of samematerial composition vary in their magnetic response with the square ofthe diameter of the bead. Therefore, a magnetic field will interactdifferentially on beads of different size allowing a sorting mechanismto take place.

There are two ways this differential magnetic response can be exploitedto enhance separation speed and specificity. As the beads leave thereservoir a magnetic field (generated through an electromagnetic force,or preferably through a permanent magnet) applied to the channel can beused to “order” the beads. What is meant by order is that since largerbeads will feel the magnetic force more strongly than smaller beads,they will move more slowly downstream than the smaller beads, resultingin a preference for smaller beads to progress down the channel earlierthan larger beads, which can decrease likelihood of bead-based cloggingof pores. Bead clogging of a pore would decrease multiplexingspecificity and could prevent proper testing altogether by restrictingthe flow of liquid needed to wash away excess enzyme and providechemical substrate for the captured enzymes to work. It is critical totune the velocity of the flow and the magnetic strength such that thelarger beads aren't captured by the magnet, preventing them flowing totheir destination sensor.

Method 2: Successively Stronger Magnetic Force for MembranelessSeparation:

Ideally, no membrane would be utilized because of the effect it has onflow rates, cost of membrane and cost of integrating membrane intocartridge. Therefore a membraneless separation technology is of extremevalue.

By exploiting the same mechanism described earlier about the magneticforce response of a bead scaling with the square of the diameter of thebead, we can achieve this separation of bead populations in a singlechannel by employing magnetic fields of successively stronger force inthe flow in close proximity to a destination sensor for each beadpopulation. In other words, instead of a series of membranes inproximity to a destination sensor, we have a series of magnetic fields(preferably set up with permanent magnets located underneath thecartridge and built into the reader) such that the largest magneticbeads are localized at the first sensor because they are unable toescape the first magnetic field, which is just strong enough to capturethe largest beads, but not strong enough to capture the second largestset of beads (or any other set of beads). Bead populations with smallerdiameters than the population with the largest beads will move ondownstream with the flow of liquid until they are caught by the magneticfield located at their intended destination sensor. The second weakestmagnetic field will capture the population of beads with the secondlargest diameter. The bead population with the third largest diameterswill be captured by the third smallest magnetic fields and so on. Thesmallest magnetic beads are caught by the strongest magnetic field.Thereby each bead population is localized to a destination sensor andthe detection proceeds as described earlier. The magnetic force willkeep the beads in place during the washing and chemical substrateaddition phase of the test.

The reference figures are FIGS. 25-26 . The top view of FIG. 25A depictsmagnets (e.g., magnet 2534), sensor 2511, sensor 2512, sensor 2513,sensor 2514, a microfluidic channel 2505, reservoirs 2501, and anabsorbent pad area 2520. The side view of FIG. 25B depicts a magnet 2531underneath sensor 2511, a magnet 2532 underneath sensor 2512, a magnet2533 underneath sensor 2513, and a magnet 2534 underneath sensor 2514.Permanent magnets immobilized in the reader (depicted as circles in topview and cylinders in side view) are order successively such thatfurther downstream of the reservoirs towards the absorbent pad, themagnetic field felt by particles flowing in the microfluidic channelgets stronger because the magnet is located at successively closer tothe sensors. This allows for the largest particles (or population ofbeads) to become magnetically localized on the sensor closest to thereservoirs (sensor 2513) while the smallest particles are localized onthe sensor furthest downstream (sensor 2514). The second largest set ofparticles end up on sensor 2512 and third largest set of particles onsensor 2513.

In FIG. 26 , a side view of the cartridge shows a magnet 2631 underneathsensor 2611, a magnet 2632 underneath sensor 2612, a magnet 2633underneath sensor 2613, and a magnet 2634 underneath sensor 2614.

Method 3: Cross-Flow Filtration Utilizing Magnetic Fields and Membranes:

Most cross flow filtration technology uses flow down a channel pluspressure and membranes perpendicular to the flow of liquid to preventmembrane fouling common with dead-end filtration. Liquid and particlessmaller than the membrane cut off size are pushed through the membraneinto another fluid channel.

The method described here uses magnetic force, instead of pressure, inorder to exert the force perpendicular (or any proper angle not directlyparallel to the flow) to the flow such that magnetic beads below acertain diameter are able to move through a membrane with pore sizelarger than its diameter towards a sensor where they will be localizedthrough magnetic force and held during the washing and detection stepsas described earlier.

This method is facilitated by use of an “aligning” or “focusing” magnetupstream of the membranes such that the magnetic beads are pulled to theside of the channel where the membranes are located such that they havethe opportunity to feel the magnetic field acting to pull beads of theright size through the membrane.

In this method, membranes with smallest pore size are most upstream andprogressively membranes with larger pore size are downstream. Otherwise,all beads smaller than the cut off would enter into the sensor regionreserved for the largest of beads.

Just as in standard crossflow filtration techniques, the two mostcritical parameters are flow velocity and transmembrane pressure (ormagnetic force in our case).

Multiple filters of the same size can be added one after another inorder to collect all beads of the proper size and it is preferable thatall of these off shoot channels collect over a single sensor but it isnot necessary.

Production of a Multiplexed Cartridge

To apply a local magnetic field in a precise manner will require aspecial cutout of the cartridge bottom such that permanent magnetsaffixed to the reader will be able to slide into place where theremagnetic field will precisely affect the beads.

Implementing the membrane placement within the cartridge duringproduction requires some innovative methods. We propose a processingstep such that the membrane is cut and adhesive applied such that thebottom (the floor of the channel) side is taped down during assembly tothe PCB sensor board. A series of vents are used such that vacuum can beapplied which will raise up the membrane such that they can form anadhesive seal against a frame on the main channel wall where themembrane will form its angled off shoot for beads smaller than themembrane pore size.

In effect, the membrane will be sucked into place and bonded throughapplied vacuum and adhesive onto the main channel's egress into the sidechannel. Membranes taped flat at the off shoots (not in the mainchannel) will be tombstoned up into place on the frame of the entranceto the side channel with applied vacuum.

Built into the cartridge are these frames, which are small protrusionsfrom the ceilings at the entrance to the side channels (offshoots).

Additionally, this principle can be applied to the dead-end filtrationmethod. Frames are built into the main channel, rather than the sidechannel, and vacuum is applied between each set of membranes and beforethe first to tombstone the membranes into place.

Although the foregoing has included detailed descriptions of someembodiments by way of illustration and example, it will be readilyapparent to those of ordinary skill in the art in light of the teachingsof these embodiments that modifications may be made without departingfrom the spirit or scope of the appended claims.

What is claimed is:
 1. A system for identifying the presence, absence,and/or quantity of a molecule of interest in a sample, comprising: areader; and a cartridge that fits into the reader, the cartridgeconfigured to accept a sample collection device that fits into thecartridge; wherein the cartridge comprises: a first reservoir forstoring a liquid, wherein a mixture comprising the liquid and the sampleis formed in the first reservoir when the sample collection devicehaving the sample is inserted into the first reservoir; a secondreservoir for storing a chemical substrate; an analysis channelcomprising an electrochemical sensor; and a first phase change valve forsealing the mixture in the first reservoir from the analysis channel insolid state; a second phase change valve for sealing the chemicalsubstrate in the second reservoir from the analysis channel in solidstate; and one or more heaters disposed adjacent the first and secondphase change valves for melting the first and second phase change valvesto transfer the mixture and the chemical substrate to the analysischannel, wherein a reaction of the mixture and the chemical substrate isdetected by the electrochemical sensor and communicated to the reader.2. The system of claim 1, wherein the mixture comprises a plurality ofmagnetic particle complexes, wherein each of the plurality of magneticparticle complexes comprises a magnetic particle.
 3. The system of claim2, wherein the reader comprises a magnetic field generator adjacent tothe analysis channel when the cartridge fits into the reader, such thatthe plurality of magnetic particle complexes can be localized in theanalysis channel within a magnetic field generated by the magnetic fieldgenerator.
 4. The system of claim 2, wherein the electrochemical sensorreleases a flow of electrons in a quantity proportional to a number ofthe plurality of magnetic particle complexes localized in the analysischannel in response to reactions between the mixture and the chemicalsubstrate.
 5. The system of claim 1, wherein the molecule of interestcomprises one or more of a nucleic acid, protein, or small molecule. 6.The system of claim 1, wherein the cartridge comprises a slot to allowfor a permanent magnet to slide into place underneath theelectrochemical sensor.
 7. The system of claim 1, wherein the reader isconfigured to send or receive, or both, signals to a mobile deviceindicative of a test result.
 8. The system of claim 7, furthercomprising an application stored on the mobile device, the applicationconfigured to cause the mobile device to display the test result.
 9. Thesystem of claim 1, wherein the reader is configured to send or receive,or both, signals to a mobile device indicative of a test protocol. 10.The system of claim 3, wherein the magnetic field generator comprises apermanent magnet.
 11. The system of claim 3, wherein the magnetic fieldgenerator comprises more than one magnet.
 12. The system of claim 1,wherein first and second phase change valves consist of wax and the oneor more heaters heat one or more vias to melt the wax.
 13. The system ofclaim 1, further comprising a sonicator configured to direct energy intothe mixture in the first reservoir.
 14. The system of claim 1, furthercomprising a third reservoir for storing a wash solution, wherein theone or more heaters facilitates transfer of the wash solution to theanalysis channel.
 15. The system of claim 1, wherein the system furthercomprises the sample collection device, the sample collection deviceconfigured to collect the sample from blood, urine, or saliva.